This research project encompasses a number of different approaches to both understand how current anti-tubercular chemotherapy works using the most modern technologies and to use this information to develop new and improved therapies and therapeutic approaches. Individual projects within this framework are; (1) understanding the activity of various drugs in animal models of tuberculosis therapy, (2) development of advanced animal models for predicting drug efficacy under conditions that exactly mimic those experienced by TB patients (3) correlating responses seen in animal models with the pathology and response to therapy observed in human TB, (4) developing structural and functional imaging techniques using CT/PET for use in live, M. tuberculosis (Mtb) infected animals, and (5) developing techniques for assessing drug distribution, penetration, and pharmacokinetics in vivo. Most of our PET-CT studies have used 18F fluorodeoxyglucose to image the metabolism of the eukaryotic cells in TB lesions but we are also making attempts to identify the location, abundance and metabolic state of the bacteria in lesions using bacteria-specific probes. Investigation of the penetration of both currently used and investigative TB drugs into Mtb rabbit lesions and human lesions is ongoing. Using non-compartmental and population pharmacokinetic approaches, we modeled the rate and extent of distribution of isoniazid, rifampicin, pyrazinamide and moxifloxacin in rabbit lung and lesions. Moxifloxacin reproducibly showed favorable partitioning into cellular regions of lesions but not the lipid-rich caseum, while the partitioning of isoniazid, rifampicin in lesions was markedly lower than in plasma. A similar study is underway in collaboration with Rutgers Univ. for pyrazinamide (in humans and other species), 5 fluoroquinolones, modified analogues of PA 824, and a number of other drugs used and being considered for use in tuberculosis. The objective of these studies is to compare the lesion penetration of similar analogues and several members of each class of drugs and use lesion penetration as a factor in the selection of better candidates for future preclinical studies. As our results suggest that increasing delivery to the site of infection may increase drug efficacy, we have been performing a series of experiments to determine if treatment with an agent that promotes normalization of blood vessel structure such that hypoxia is decreased and drug penetration is increased could improve drug access to the lesion. In collaboration with Mass General Hospital we have determined that TB lesion-associated vasculature is structurally and functionally abnormal. Treatment with the normalization agent yielded transient increases in small molecule penetration and oxygenation of the TB lesions. Attempts to normalize the vasculature of lesions and maintain that normalization for a relevant duration to treatment of tuberculosis will continue, but we are also agents that could modify the structure of the TB lesion at various stages of lesion development and in lesions being treated with combination therapy. These experiments are ongoing in the rabbit model with results monitored by FDG-PET/CT imaging, lesion histology, drug quantification and bacterial load. We have continued developing a new, non-human primate (NHP) model for tuberculosis - the common marmoset. To establish the susceptibility of this new world NHP to developing TB, we aerosol infected marmosets with one of three Mtb complex strains of diverse pathogenic potential. All three strains also resulted in pathology at necropsy that was highly similar to that observed in human TB patients. Quantitative assessment of disease burden by FDG-PET/CT allowed an accurate assessment of disease progression in these animals that was highly correlated with pathology findings at necropsy. Encouraged by these results, we began exploring if the marmoset model accurately reflects the response to treatment by providing standard TB treatment (RIF, INH, PZA, and EMB) to infected symptomatic marmosets. We compared the sterilizing potential of two regimens known to differ in sterilizing potential by virtue of very different relapse rates observed in human clinical trials. Both regimens resulted in comparable clearance of bacilli from total lung and granulomatous lesions after two months of treatment and reduction in size of individual lesions correlated with lower bacterial burden at necropsy. Within 2 weeks to 1 month of treatment, CT and FDG-PET were both able to distinguish the differing effectiveness of these two regimens when applied serially to treated animals suggesting more marked early reduction in overall metabolic activity in the lungs (decreased FDG uptake) and more rapid change in dense disease volume by CT is associated with effective treatment. The known association of cavitary disease with disease relapse suggests that this small non-human primate model of infection provides an early evaluation of the sterilizing potential of new antitubercular regimens, offering a powerful new methodology for prioritizing new combinations. These results support our previous results in the rabbit model suggesting that PET/CT may be an important early correlate of efficacy of novel combinations of new drugs that can be directly translated to human clinical trials.